Kyongmin Kim

1. Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea2. Department of Biomedical Science, Graduate School of Ajou University, Suwon, Korea3. Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea

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Abstract

Among the seven serines and one threonine in the carboxyl-terminus of HBV C protein, all but one (serine 183) appear in the context of RxxS/T consensus phosphoacceptor motifs and also overlap with other consensus motifs, such as S/TP, RS, SPRRR, RRRS/T, or RRxS/T, suggesting that various cellular kinases phosphorylate these residues. To determine whether threonine and/or serine (serines 157, 164, 170, 172, 178, and 180, and threonine 162, adw subtype) of HBV C protein are indeed phosphoacceptor residues in cells, Huh7 were transfected with a series of C-protein-expressing mutants, labeled with 32P-orthophosphate for 14 h, and then lysed. The 32Pi-labeled lysates were immunoprecipitated with anti-HBc antibody, and the 32Pi-labeled immunoprecipitated C proteins were detected by autoradiography.

For transfections, 10 µg of plasmid encoding HBV STSSSS (WT) or phosphoacceptor-site mutant C proteins in a P-deficient mutant backbone were used. As a transfection control, 1 µg of green fluorescent protein (GFP)-expressing plasmid was included in the transfection mixture.

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